Evaluation of direct versus multi-layer passivation and capture chemistries for nanoparticle-based biosensor applications.

Nanoparticles used in biosensor applications often fail when deployed directly in complex biological fluids. This is due to surface fouling and interference from the large concentration of non-specific binders (proteins, lipids, nucleic acids and saccharides) in the matrix. We systematically investigate four orthogonal approaches for decorating nanoparticle surfaces with affinity probes and evaluate their performance in buffer and serum. Carbodiimide coupling, cooper-mediated 'click' coupling, copper-free click coupling and thiol-maleimide coupling were quantitatively controlled during the fabrication process. Analyte mediated aggregation of fluorescent reporters and paramagnetic nanoparticle in a sandwich immunoassay was then used to probe assay sensitivity and specificity using an early biomarker of dengue fever, NS-1, as an exemplar and clinically relevant analyte. The type of surface functionalization played a vital role in assay performance in buffer versus serum at the assay sensitivity limit (3 ng/mL in serum) and over the linearity of response of the assay's dynamic range. There was a 10 fold increase on the dynamic range of the detection of NS1 comparing copper free click coupling to carbodiimide coupling, one of the most common approaches for nanoparticle functionalization. By tuning their size, we could carefully monitor the evolution of nanoparticle populations by flow cytometer and discriminate between unbound and fluorescent nanoparticles. This subtle control on each assay component resulted in more than a 10-fold reduction in fluorescence background and improved the sensitivity of almost two orders of magnitude compared to endpoint measurements. (C) 2014 Elsevier B.V. All rights reserved.